5-methoxytryptophan and its derivatives and uses thereof

ABSTRACT

A 5-methoxytryptophan and its derivatives are disclosed, wherein the 5-methoxytryptophan and its derivatives are represented by the following formula (I): 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , and n are defined in the specification. In addition, the present invention also provides novel used of the 5-methoxytryptophan and its derivatives for treating inflammatory-related disease and cancers.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to 5-methoxytryptophan and its derivativesand uses thereof. More specifically, the present invention relates tonovel 5-methoxytryptophan and its derivatives and uses thereof fortreating inflammatory-related diseases and cancers.

2. Description of Related Art

Abnormal activation of innate immune system has been implicated in thedevelopment of inflammatory disorders such as septic shock, multipleorgan failure and atherosclerosis. Toll-like receptors (TLRs) play acritical role in regulating immune response and maintaining immunehomeostasis. Inappropriate activation of TLR signaling by pathogencomponents and endogenous harmful molecules is a major contributor tosystemic inflammation such as sepsis. There is growing evidence thatTLRs play a key role in mediating systemic responses to invadingpathogens during systemic inflammation and sepsis. In particular,activation of TLR4 by LPS is thought to be an important trigger ofinflammatory response in sepsis. In addition to LPS, TLR4 can also beactivated by endogenous molecules such as high mobility group box 1(HMGB1) as a late mediator of lethal sepsis, which in turn initiates asecondary immunostimulatory cascade. Systemic inflammatory responsesyndrome is induced most commonly by a systemic infection ofgrain-negative bacteria and the subsequent release of LPS, whichactivates TLR4 signaling. The excessive stimulation of the host immunesystem by LPS results in high levels of inflammatory cytokines in thecirculation and disseminated intravascular coagulation.

Systemic inflammation is characterized by metabolic syndrome,cardiovascular decompensation, multiple organ failure, disseminatedintravascular coagulation and shock. These clinical manifestations areattributed to inappropriate or extensive inflammatory responses touncontained bacterial infection and the endotoxins produced bygrain-negative bacteria notably lipopolysaccharide (LPS). Excessiveimmune responses lead to production of very high levels ofproinflammatory cytokines (cytokine storm) and overexpression ofproinflammatory mediators such as cyclooxygenese-2 (COX-2) and induciblenitric oxide synthase (iNOS). Treatment of systemic inflammatoryresponse syndrome is limited to fluid administration, oxygen supplement,antibiotics and other supportive measures. Specific therapeuticapproaches targeting proinflammatory cytokines, coagulation cascade, LPSor immune responses are unsuccessful or have marginal efficacy.

Abnormal activation of the innate immune system and the resultingchronic inflammation have been implicated in the development andprogression of chronic and metabolic diseases including atherosclerosis(Hansson G K & Hermansson A. The immune system in atherosclerosis. NatImmunol 12:204-212 (2011)). Considerable evidence suggests thatinfectious agents could contribute to cardiovascular diseases. Vascularinjury are often caused by uncontrolled infection with releases ofendotoxins notably LPS. LPS activates toll-like receptor 4 (TLR4) whichtransmits signals to activate NF-□B and C/EBP□ resulting in excessiveproduction of proinflammatory mediators thereby inducing endothelial andvascular damages. In addition to LPS, TLR4 can also be activated byendogenous molecules such as high mobility group box 1 (HMGB1) as a latemediator of inflammation, which contributes to the development ofatherosclerosis. ApoE-knockout mouse challenged with Porphyromonasgingivalis not only accelerates atherosclerosis but also increasesexpression of TLR2 and TLR4. Vascular disease is recognized as aninflammatory disease—inflammation activates endothelium and theunderlying medial VSMCs (Hansson G K. Inflammation, atherosclerosis, andcoronary artery disease. N Engl J Med 352, 1685-95 (2005)). Thepathogenic VSMC migration and proliferation (leading toarteriosclerosis) are somewhat similar to that of systemic inflammationas well as cancer cell growth and invasion. We hypothesized that 5-MTPmight protect against vascular injury-induced inflammation and thesubsequent endothelial dysfunction and VSMC proliferation and migration,and consequent neointima formation.

Therefore, it is desirable to provide a new therapeutic agents andmethods to conquer the aforementioned serious human illness.

SUMMARY OF THE INVENTION

This invention is based on the discovery that certain5-methoxytryptophan and its derivatives can be used as anti-inflammatorydiseases and anti-cancer agents. Thus, the object of the presentinvention is to provide 5-methoxytryptophan and its derivatives andmethods for treating an inflammatory-related disease and cancers withthe same.

One aspect of the present invention is related to a compound of formula(I):

In this formula, R₁ is H, halogen atom, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₂ is C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀cycloalkenyl; R₃ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₄ is H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl,C(O)R_(a), or C(O)OR_(a); in which R_(a) is H, C₁-C₁₀ alkyl, C₃-C₂₀cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; R₅ is H,C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, orheteroaryl; and n is 0-5.

One subset of the just-described compounds includes 5-methoxytryptophanand its derivatives, in which each R₁ and R₂, independently, is H orC₁-C₁₀ alkyl. In these compounds, preferably, R₁ is H, and R₂ is C₁-C₃alkyl.

Another subset of the just-described compounds includes5-methoxytryptophan and its derivatives, in which R₃ is H, C₁-C₁₀ alkyl,and R₄ is H, C₁-C₁₀ alkyl, C(O)R_(a), or C(O)OR_(a), in which R_(a) isH, or C₁-C₁₀ alkyl. In these compounds, preferably, R₃ is H, and R₄ is Hor C(O)R_(a), in which R_(a) is C₁-C₃ alkyl.

Anther subset of the just-described compounds includes5-methoxytryptophan and its derivatives, in which R₅ is H or C₁-C₁₀alkyl. In these compounds, preferably, R₅ is H or C₁-C₃ alkyl.

A further subset of the just-described compounds includes5-methoxytryptophan and its derivatives, in which n is 1 or 2.

The term “alkyl” refers to a saturated, linear or branched hydrocarbonmoiety, such as —CH₃ or —CH(CH₃)₂. Examples of alkyl include, but arenot limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, andt-butyl. The term “alkenyl” refers to a linear or branched hydrocarbonmoiety that contains at least one double bond, such as —CH═CH—CH₃.Examples of alkenyl include, but are not limited to, ethenyl, propenyl,propenylene, allyl, and 1,4-butadienyl. The term “alkynyl” refers to alinear or branched hydrocarbon moiety that contains at least one triplebond, such as —C≡C—CH₃. Examples of alkynyl include, but are not limitedto, ethynyl, ethynylene, 1-propynyl, 1- and 2-butynyl, and1-methyl-2-butynyl. The term “cycloalkyl” refers to a saturated, cyclichydrocarbon moiety. Examples of cycloalkyl include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cycloheptyl, and cyclooctyl. The term “cycloalkenyl”refers to a non-aromatic, cyclic hydrocarbon moiety that contains atleast one double bond, such as cyclohexenyl. The term “heterocycloalkyl”refers to a saturated, cyclic moiety having at least one ring heteroatom(e.g., N, O, or S). Examples of heterocycloalkyl groups include, but arenot limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, andtetrahydrofuranyl. The term “aryl” refers to a hydrocarbon moiety havingone or more aromatic rings. Examples of aryl moieties include phenyl(Ph), phenylene, naphthyl, naphthylene, pyrenyl, anthryl, andphenanthryl. The term “heteroaryl” refers to a moiety having one or morearomatic rings that contain at least one heteroatom (e.g., N, O, or S).Examples of heteroaryl moieties include furyl, furylene, fluorenyl,pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl,pyrimidinyl, quinazolinyl, quinolyl, isoquinolyl and indolyl. The term“halogen atom” refers to F, Cl, Br or I.

Alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,aryl, and heteroaryl mentioned herein include both substituted andunsubstituted moieties, unless specified otherwise. Possiblesubstituents on cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, andheteroaryl include, but are not limited to, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, C₁-C₂₀heterocycloalkyl, C₁-C₂₀ heterocycloalkenyl, C₁-C₁₀ alkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, amino, C₁-C₁₀ alkylamino, C₁-C₂₀dialkylamino, arylamino, diarylamino, C₁-C₁₀ alkyl sulfonamino, arylsulfonamino, C₁-C₁₀ alkylimino, arylimino, C₁-C₁₀ alkylsulfonimino,arylsulfonimino, hydroxyl, halo, thio, C₁-C₁₀ alkylthio, arylthio,C₁-C₁₀ alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl,amido, amidino, guanidine, ureido, thioureido, cyano, nitro, nitroso,azido, acyl, thioacyl, acyloxy, carboxyl, and carboxylic ester. On theother hand, possible substituents on alkyl, alkenyl, or alkynyl includeall of the above-recited substituents except C₁-C₁₀ alkyl. Cycloalkyl,cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroarylcan also be fused with each other.

The compounds described above include the compounds themselves, as wellas their salts, their solvates, and their prodrugs, if applicable. Asalt, for example, can be formed between an anion and a positivelycharged group (e.g., amino) on 5-methoxytryptophan and its derivatives.Suitable anions include chloride, bromide, iodide, sulfate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate,tosylate, tartrate, fumurate, glutamate, glucuronate, lactate,glutarate, and maleate. Likewise, a salt can also be formed between acation and a negatively charged group (e.g., carboxylate) on5-methoxytryptophan and its derivatives. Suitable cations include sodiumion, potassium ion, magnesium ion, calcium ion, and an ammonium cationsuch as tetramethylammonium ion. The compounds also include those saltscontaining quaternary nitrogen atoms. Examples of prodrugs includeesters and other pharmaceutically acceptable derivatives, which, uponadministration to a subject, are capable of providing active compoundsas described above. A solvate refers to a complex formed between anactive 5-methoxytryptophan or its derivative and a pharmaceuticallyacceptable solvent. Examples of pharmaceutically acceptable solventsinclude water, ethanol, isopropanol, ethyl acetate, acetic acid, andethanolamine.

One subset of the just-described compounds includes 5-methoxytryptophanderivatives, with the proviso that R₁, R₃, R₄ and R₅ are not H when R₂is C₁-C₁₀ alkyl.

In another aspect, this invention features a method for treating aninflammatory-related disease. The method includes administering to asubject in need thereof and effective amount of one or more compounds offormula (I) shown above. Examples of inflammatory-related diseasesinclude sepsis, Systemic Lupus Erythematosus (SLE), cardiovasculardiseases, metabolic syndrome, cancer, septicemia and diverseinflammatory joint, gastrointestinal, and renal diseases.

In further another aspect, this invention also features a method fortreating a cancer. The method includes administering to a subject inneed thereof and effective amount of one or more compounds of formula(I) shown above. Cancer that can be treated by the method of thisinvention includes both solid and haematological tumours of variousorgans. Examples of solid tumors include pancreatic cancer, bladdercancer (e.g., urothelium cancer), colorectal cancer, breast cancer(e.g., metastatic breast cancer), male genital tract cancer (e.g.,seminal vesicle cancer, testes cancer, germ cell tumors, and prostatecancer such as androgen-dependent and androgen-independent prostatecancer), renal cancer (e.g., metastatic renal cell carcinoma),hepatocellular cancer, lung cancer (e.g., small cell lung cancer,non-small cell lung cancer, bronchioloalveolar carcinoma, andadenocarcinoma of the lung), ovarian cancer (e.g., progressiveepithelial or primary peritoneal cancer), cervical cancer, uteruscancer, gestational trophoblastic disease (e.g., choriocarcinoma),gastric cancer, bile duct cancer, gallbladder cancer, small intestinecancer, esophageal cancer, oropharyngeal cancer, hypopharyngeal cancer,eye cancer (e.g., retinoblastoma), nerve cancer (e.g., Schwannoma,meningioma, neuroblastoma, and neuroma), head and neck cancer (e.g.,squamous cell carcinoma of the head and neck), melanoma, plasmacytoma,endocrine gland neoplasm (e.g., pituitary adenoma, thyroid cancer, andadrenal tumor), neuroendocrine cancer (e.g., metastatic neuroendocrinetumors), brain tumors (e.g., glioma, anaplastic oligodendroglioma,glioblastoma multiforme, and astrocytoma such as adult anaplasticastrocytoma), bone cancer, and sarcomas from soft tissue or bone (e.g.,Kaposi's sarcoma). Examples of hematologic malignancy include acutemyeloid leukemia, chloroma, chronic myelogenous leukemia or CML (e.g.,accelerated CML and CIVIL blast phase), acute lymphoblastic leukemia,chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma(e.g., follicular lymphoma, cutaneous T-cell lymphoma such as mycosisfungoides, and mantle cell lymphoma), B-cell lymphoma, multiple myeloma,Waldenstrom's macroglobulinemia, myelodysplastic syndromes (e.g.,refractory anemia, refractory anemia with ringed siderblasts, refractoryanemia with excess blasts or RAEB, and RAEB in transformation or RAEB-T,and myeloproliferative syndromes).

The term “treating” or refers to administering one or more5-methoxytryptophan and its derivatives to a subject, who has anabove-described disease, a symptom of such a disease, or apredisposition toward such a disease, with the purpose to confer atherapeutic effect, e.g., to cure, relieve, alter, affect, ameliorate,or prevent the above-described disease, the symptom of it, or thepredisposition toward it.

The term “an effective amount” refers to the amount of a5-methoxytryptophan or its derivative that is required to confer atherapeutic effect on the treated subject. Effective amounts may vary,as recognized by those skilled in the art, depending on the types ofdiseases treated, route of administration, excipient usage, and thepossibility of co-usage with other agents.

Also within the scope of this invention is a pharmaceutical compositioncontaining one or more 5-methoxytryptophan and its derivatives describedabove for use in treating one of the above-described diseases andcancers, and the use of such a composition for the manufacture of amedicament for this treatment.

Shown below are exemplary 5-methoxytryptophan and its derivatives of thepresent invention:

wherein 5-MTP refers to 5-methoxytryptophan; 5-MTPE refers toester-5-MTP; and NACT-5MTP refers to N-acetyl-5-MTP.

Other objects, advantages, and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows structures of 5-MTP and its derivatives of the presentinvention.

FIG. 1B shows thin layer chromatography and mass spectra of 5-MTPE.

FIGS. 1C-1E show HNMR spectra of 5-MTP and its derivatives (5-MTPE andNACT-5MTP).

FIG. 1F shows results of suppressive activity of 5-MTP and itsderivatives in PMA-induced A549 COX-2 expression.

FIG. 2A shows results of cell migration assays in A549 lung cancer cellsby treating 5-MTP or 5-MTPE.

FIG. 2B shows results of cell migration assays in BT474 and T47D breastcancer cells by treating 5-MTPE.

FIG. 2C shows results of tumor xenograft experiment by treating 5-MTP or5-MTPE, wherein * denotes p<0.05, and ** donates p<0.01.

FIGS. 3A-3F respectively show inhibitions of 5-MTP on LPS-inducedexpression of COX-2 and different cytokines.

FIGS. 4A-4B shows effect of 5-MTPE on cell viability and IL-6production.

FIG. 5A shows survival of mice injected with and without 5-MTP or 5-MTPEbefore these animals were challenged with LPS.

FIG. 5B shows survival of mice injected with and without 5-MTP 30 minsafter cecal ligation and puncture (CLP) surgery.

FIG. 5C shows inhibitions of 5-MTP on mice challenged with LPS.

FIG. 5D shows results of bronchoalveolar lavage fluid (BALF).

FIGS. 6A-6B show inhibitions of 5-MTP on LPS-induced expression of COX-2and iNOS.

FIGS. 7A-7D respectively show different proinflammatory cytokines levelsin mice treated with or without 5-MTP.

FIG. 7E shows proinflammatory cytokines levels in peritoneal macrophagestreated with or without 5-MTP.

FIG. 7F shows serum 5-MTP levels in mice treated with saline or LPS withor without 5-MTP.

FIG. 8A shows inhibitions of 5-MTP on neutrophil infiltration.

FIG. 8B shows MPO activity in lung tissues of mice treated with salineor LPS with or without 5-MTP.

FIG. 8C shows serum chemokine levels in mice treated with saline or LPSwith or without 5-MTP.

FIG. 9A shows activated caspase-3 level in lung tissues of mice treatedwith saline or LPS with or without 5-MTP.

FIG. 9B shows activated caspase-3 level in splenocytes of mice treatedwith saline or LPS with or without 5-MTP.

FIG. 9C shows spleen weight/body weight ratio of LPS induced-micetreated with or without 5-MTP.

FIG. 10A shows immunoblotted results of p38, phospho-p38 (p-p38),ERK1/2, phospho-ERK1/2 (p-ERK1/2) or β-actin in RAW264.7 cells treatedwith or without 5-MTP.

FIG. 10B shows NF-κB promoter activity in RAW264.7 cells treated with orwithout 5-MTP.

FIG. 10C shows immunoblotted results of NF-κB p65, phospho-NF-κB p65(p-p65) (Ser536) or β-actin in RAW264.7 cells treated with or without5-MTP.

FIG. 10D shows phosphor-p65 level in the lysates of peritonealmacrophages treated with LPS with or without 5-MTP.

FIG. 10E shows phosphor-p65 level in lung tissues of mice after LPSinfusion with or without 5-MTP.

FIG. 10F shows p300 HAT activity in peritoneal macrophages treated withLPS in the presence or absence of 5-MTP.

FIG. 10G shows p300 HAT activity in lung tissues of mice after LPSinfusion with or without 5-MTP.

FIG. 10H shows schematic illustration of the signaling mechanisms viawhich 5-MTP inhibits systemic inflammatory syndrome.

FIGS. 11A-11B shows IL-6 levels in culture supernatants of RAW264.7cells treated with 5-MTP, 5-MTPE, COX-2 inhibitors (NSC398 or SC560) andNF-κB inhibitor JSH-23 after LPS treatment.

FIG. 11C shows luciferase activity after RAW264.7 cells were transfectedwith COX-2 promoter-luciferase plasmid.

FIG. 12A shows serum 5-MTP concentrations in patients with coronaryartery disease (CAD) (n=50) and healthy subjects (n=30) were measured bycompetitive 5-MTP ELISA.

FIG. 12B shows inhibition of 5-MTP on lipid accumulation andatherosclerotic lesion formation in arteries of ApoE-deficient micetreat with high fat diet.

FIG. 12C shows inhibitions of 5-MTPE on lipid accumulation inatherosclerotic lesions of ApoE-deficient mice treat with high fat diet.

FIG. 12D shows suppressive effects of 5-MTP and 5-MTPE on calcifyingmedium-induced calcification in vascular smooth muscle cells.

FIG. 12E shows inhibitions of 5-MTP on high fat diet-induced vascularcalcification in ApoE-deficient mice.

FIG. 13 shows attenuation of 5-MTP on intimal hyperplasia of the ligatedmouse carotid arteries. Vessel sections were stained with H&E (A, B) orVerhoeff's staining for elastin (black) (C, D). (E) Quantitativemorphometric analysis of intimal area in the ligated carotid arteries.Compared with vehicle-treated mice (0.96±0.19, n=9), 5-MTP reducedintima/media area ratio to 0.58±0.11 (n=12; *P<0.05 vs. vehicle group).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT [Synthesis of 5-MTP andits Derivatives]

Synthesized 5-MTP provided by ASTATECH (PA, USA) was acted as materialfor 5-MTP derivatives synthesis as shown in synthetic scheme; and thestructures of 5-MTP and its derivatives are shown in FIG. 1A.

Experimental Procedures Synthetic Step 1:

At 10° C., 33% Aq. Dimethylamine was carefully added dropwise to AcOH,and while controlled the inner temperature below 20° C., 40% Aq.Formaldehyde was added slowly.

To above solution at 10° C., 5-methoxyindole was added portionwise, andstirred under 30° C. overnight. TLC showed the starting material5-methoxyindole was consumed. At 10° C., adjusted to pH>9 with 1N NaOHaq., extracted with MTBE five times, combined organic phases was washedwith brine, dried over Na2SO4, concentrated to afford a red oil, washedwith Petroleum ether got an off-white solid Compound 1 (250.0 g, yield:75.2%).

Synthetic Step 2:

Compound 1 and 2-acetylaminomalonic acid diethyl ester were dissolved intoluene, solid NaOH was then added, and heated reflux for 18 h. TLCchecked. Cooled to 10° C. while stirring, and stood for 2 h. Collectedthe precipitate by filtration, dried to afford a white solid Compound 2(180.0 g, yield 98%).

Synthetic Step 3:

Compound 2 in 2N NaOH were heated to reflux for 3 h, TLC checked. Cooledto R.T., acidified to pH=2, extracted with EA for five times, combinedorganic phases was washed with brine, dried over Na₂SO₄, concentrated toafford a white solid Compound 3 (150.0 g, Yield:100%).

Synthetic Step 4:

Compound 3 in water was heated reflux for 3 hrs, TLC checked. Cooled toR.T., Collected the precipitate by filtration, dried to afford a whitesolid Compound 4 (98.0 g, yield 76%).

Synthetic Step 5 (Synthesis of 5-MTP):

Compound 4 was added to a solution KH₂PO₄ (50 mM) in water, KOH wasadded stirred for 10 min., got a clean solution. CoCl₂ (50 mM) wasadded, keep pH=8, used KOH (2N aq), Acylase was added and 35-40° C.reacted for 16 hrs, TLC checked. The precipitate was collected byfiltration, washed with water until the Purification of EE % above 98%,dried to afford 5-MTP (14.0 g product. Yield, 33%).

Synthetic Step 6 (Synthesis of 5-MTPE):

5-MTP was dissolved in MeOH with catalytic amount of Conc.H₂SO₄, thesolution was then heated reflux for 24 hrs, TLC checked. The MeOH wasconcentrated and the residue was poured in water and basified to ph=7.5with NaHCO₃, extracted with DCM for three times, combined organic phaseswashed with brine, dried over Na₂SO₄, concentrated to afford a red oil,crystallized in MTBE to afford brown solid 4.8 g of 5-MTPE (Yield:77%).

SYNTHETIC Step 7 (Synthesis of NACT-5MTP):

1 g of the 5-MTP was dissolved in a cooled (2N) NaOH 50 mL, At 0° C. orlower, 0.5 mL Ac₂O was added dropwise, inner temperature was below 0°C., then stirred in the same condition for 0.5 hrs, acidified to pH=2,The precipitate was collected by filtration, washed with water and dry24h at 50° C. to get 1 g NACT-5MTPE (cat#22099).

The purity and chemical structure of the obtained 5-MTPE and NACT-5MTPwere determined by thin layer chromatography (TLC), LC-MSMS and photonNMR, respectively, and these results are shown in FIGS. 1B-1F.

[In Vitro and In Vivo Experiments] Cell Culture and Treatment

Mouse RAW264.7 macrophages were from the American Type CultureCollection and cultured in DMEM containing 10% FBS. Cells were typicallypre-incubated with or without 5-MTP (Sigma-Aldrich) for 30 min beforeLPS treatment, unless specified otherwise. The duration of LPS (100ng/ml) treatment varied depending on the experiment.

For peritoneal macrophage isolation, C57BL/6J mice were injectedintraperitoneally with 2 ml of 4% thioglycollate. Four days afterinjection, the peritoneal cavity was washed with 5 ml of ice-coldRPMI1640 medium, and cells from the peritoneal exudates were collectedby centrifugation, and suspended in RPMI1640 medium, then seeded on15-cm dishes and allowed to adhere for 4 h. Floating cells were washedout, and adherent cells were used as peritoneal macrophages in theexperiments. The cells were incubated with or without LPS (100 ng/ml)for the indicated time before each experiment, as in the protocol forthe RAW264.7 cells.

Cell Migration and Invasion Assays

Briefly, cells (10⁴/well) were seeded on the upper chamber (6.5-mminsert, 8-μm polycarbonate membrane; Corning Inc.), which was placed ineach well of a 24-well plate. The lower chamber was filled with 700 μLDMEM supplemented with 10% FBS. For assessment of in vitro invasion,cells were seeded onto the upper chamber insert coated with Matrigel (BDBiosciences) under the same conditions as the transwell migrationassays. At the indicated time, nonmigrated cells that remained at thetop surface of the insert were removed with a cotton swab. Cells thatmigrated to the lower membrane surface and cells that invaded throughthe matrigel gel to the underside of the membrane were fixed, stainedwith 0.1% crystal violet for 20 min, and counted under light microscopy.All of the assays were done in triplicate with excellentreproducibility.

Tumor Xenograft Experiment

Six-week-old male CB-17 SCID mice, purchased from BioLASCO, were housedin a daily cycle of 12-h light and 12-h darkness and pathogen-freeconditions at 26° C. at the Animal Center of the National HealthResearch Institutes. A549-luc-C8 cells (Caliper Life Sciences), culturedin RPMI-1640 supplemented with 10% FBS, were pretreated with 100 μM5-MTP, 100 μM 5-MTPE or vehicle for 5 h and were inoculated into theflank of mice subcutaneously (s.c.) (5×10⁶ cells/mouse). The 5-MTP-, or5-MTPE-treated mice (n=10) were injected with 100 mg 5-MTP or 5-MTPE/kgbody weight intraperitoneally twice weekly. The vehicle group (n=10) wasinjected with vehicle (0.033 N HCl in RPMI medium, neutralized withNaOH) twice weekly. The size of s.c. tumors were caliper-measured twiceweekly. The tumor volume was calculated according to the formula oflength×width×width/2. The tumor growth was also monitored weekly by anIVIS spectrum imaging system. Mice were euthanized at day 52.Subcutaneous tumors and lungs were removed and fixed with 10% Formalin.Nodules on each lobe of the lungs were counted. Histology of the noduleswas examined under microscopy by tissue section and H&E staining. Theanimals' care was in accordance with institutional guidelines and theprotocol of in vivo experiments was approved by the Institutional AnimalCare and Utilization Committee of the National Health ResearchInstitutes.

Induction of Endotoxemia in a Mouse Model

C57BL/6 mice (6-8 wks old) were treated intraperitoneally with saline orwith different concentrations of 5-MTP (23.4 or 100 mg/kg) for 30 minbefore intraperitoneal administration of LPS (60 mg/kg). Animals weremonitored for survival and other clinical signs including ruffled fur,lethargy, diarrhea, and body weight loss. Some animals were sacrificedat different times after LPS injection. Blood samples, peritonealexudates, lungs, and spleens were collected. All mouse experiments wereapproved by the Institutional Animal Care and Use Committee, NationalHealth Research Institutes.

Cytokine-Specific ELISA

Cytokine levels in the culture supernatants and serum were determined inmicrotiter plates (96-well) by a specific sandwich ELISA (Biosource) aspreviously described (Wu, J. Y. & Kuo, C. C. Pivotal role ofADP-ribosylation factor 6 in Toll-like receptor 9-mediated immunesignaling. J Biol Chem 287, 4323-4334 (2012)) (Lee, G L., et al. TLR 2induces vascular smooth muscle cell migration through cAMP responseelement-binding protein-mediated interleukin-6 production. ArteriosclerThromb Vasc Biol 32, 2751-2760 (2012); hereinafter, Lee, G L., et al.).

Western Blot Analysis

Cellular protein were resolved by 5% to 20% SDS-PAGE and transferred tonitrocellulose membranes and blotted with specific antibodies aspreviously described (Lee, G L., et al.).

Promoter-Luciferase Reporter Assay

RAW264.7 macrophages were cotransfected with COX-2 promoter-luciferase,IL-6 promoter-luciferase constructs as previously described (Lee, G L.,et al.) or p5xNF-κB-luciferase (Stratagene) and pcDNA3.1-β-galactosidaseplasmids using FuGENE 6 (Roche). After overnight transfection, the cellswere incubated with or without LPS in the presence or absence of 5-MTPfor 8 h. After treatment, cells were lysed, and luciferase activity wasmeasured using an assay kit (Promega). β-galactosidase activity was usedto normalize the data.

MPO Activity Assay

Myeloperoxidase (MPO) activity of lung tissue was assayed byMyeloperoxidase fluorometric detection kit (Enzo Life Sciences). Inbrief, lung tissue specimens were homogenized by Tissuelyser II (Qiagen)in tissue protein extraction buffer (pH 7.4 25 mM Tris buffer, 150 mMNaCl, 0.5% sodium deoxycholate, 2% NP-40 and 0.2% SDS), and centrifugedat 12,000×g for 20 min at 4° C. The supernatants were removed and thepellets were homogenized by Tissuelyser II for 30 seconds in 1 ml assaybuffer with 0.5% hexadecyltrimethylammonium, and then samples werefrozen, thawed three times, and centrifuged at 8,000×g for 20 min.Collected supernatants and standard were mixed with reaction cocktail(50 μM detection reagent and 20 μM hydrogen peroxide in 1× assay bufferat room temperature in the dark. After 30 min incubation, MPO activitywas determined by measuring the fluorescence intensity at excitation 530nm and emission at 600 nm in a fluorescent plate reader.

Caspase-3 Activity Assay

Caspase-3 activity was determined by the cleavage of the fluorometricsubstrate z-DEVD-AMC (Upstate Biotechnology) as previously described(Kuo, C. C., Liang, C. M., Lai, C. Y. & Liang, S. M. Involvement of heatshock protein(Hsp)90 beta but not Hsp90 alpha in antiapoptotic effect ofCpG-B oligodeoxynucleotide. J Immunol 178, 6100-6108 (2007)).

Histology and Immunohistochemistry

For histological studies, the lungs were perfused with saline, andimmersed in formalin for 24 h. Tissue blocks were placed in formalin,dehydrated in a graded series of ethanol, embedded in paraffin, cut into3 μm-thick serial sections, and stained with haematoxylin and eosin fordetecting inflammatory cells, alveolar congestion and alveolar septalwall thickness and interstitial edema.

Prior to immunohistochemical detection of COX-2, iNOS, Ly6g, or cleavedcaspase-3 in lung sections, sections were deparaffinized with xylene,progressively rehydrated through graded alcohols. Antigen sites wereretrieved by heating the sections on slides in pH 8 EDTA antigenretrieval (Trilogy; Cell Marque Corporation) in electric pressure cookerfor 15 min. Sections were sequentially blocked by UltraVision hydrogenperoxide block (Thermo) for 10 minutes and UltraVision protein block foradditional 5 minutes. All antibodies described hereafter were diluted inblocking buffer. Sections were incubated at room temperature for 2 hwith primary antibody and then washed in PBST. The sections wereincubated with primary antibody amplifier quanto (Thermo) for 10minutes. After rinsing with PBST, the sections were incubated with HRPPolymer Quanto (Thermo) for 10 minutes and washed three times with PBST.COX-2, iNOS, Ly6g and cleaved caspase-3 were visualized by the additionof DAB Quanto Chromogen: Substrate for 3 minutes. Tissues were alsocounterstained with hematoxylin.

Patient Enrollment and Measurement of Serum 5-MTP Levels

Forty patients with CAD (161 men and 66 women; mean age, 61.2±12.13years) and 80 control subjects (43 men and 37 women; mean age, 40±13.34years) without CAD and known systemic disease were enrolled in the studybetween September 2013 and July 2015. The Ethics Committee on HumanStudies at Tri-Service General Hospital, National Defense Medical Centerin Taiwan approved the study protocol (TSGHIRB #2-102-05-104 and2-102-05-105) and written informed consent was obtained from allparticipants. The presence of CAD was confirmed by coronary angiographyand CAD was defined as more than 50% angiographic diameter stenosis inone or more coronary arteries. Blood was drawn from patients prior to 23angiography and serum collected and stored at −80° C. until analysis.

Lipid Accumulation and Atherosclerotic Lesion Formation in Arteries ofMice

ApoE-deficient mice at 6 weeks were placed on a Western high fat dietcontaining 1.25% cholesterol and 21% fat (Research Diets). The mice weresimultaneously treated with vehicle (PBS), 5-MTP or 5-MTPE (23.4 mg/kgbody weight, 3 times a week) by intraperitoneal injection. After 8weeks, the aortic trees were dissected and lesions examined in theaortic arch and its branches. The aortas were carefully freed ofconnective and adipose tissue under a dissection microscope, openedlongitudinally and stained with Oil red O.

Arterial Calcification in Mice

ApoE-deficient mice were fed with on a Western high fat diet containing1.25% cholesterol and 21% fat (Research Diets) starting at 6 weeks ofage. The mice were simultaneously treated with vehicle (PBS) or 5-MTP(23.4 mg/kg body weight, 3 times a week) by intraperitoneal injection.After 20 weeks, the aortic trees were dissected and lesions examined inthe aortic arch and its branches. Vascular calcifications in the aorticroot were evaluated by von Kossa staining.

Neointima Formation Model of Carotid Artery Ligation in Mice

Approximately 12-week-old male C57BL/6 wild-type mice (NationalLaboratory Animal Center, Taiwan) were subjected to a neointimaformation model by ligating the left common carotid artery (LCCA). TheInstitutional Animal Care and Use Committee of National Health ResearchInstitutes, Taiwan approved all experimental procedures(#NHRMACUC-101144-A). Mice were anesthetized with tribromoethanolsolution at a dose of 250 mg/kg by IP injection. After assessing thelevel of anesthesia by checking the pedal reflex, the LCCA was exposedthrough a midline incision in the neck. The LCCA was then ligated nearthe carotid bifurcation with a suture, skin closed, and the animals wereallowed to recover from anesthesia and showed no symptoms of stroke.Following surgery, mice were treated with vehicle (PBS) or 100 mg/kg of5-MTP (Sigma, M4001) by IP injection 3 times a week. 5-MTP was dissolvedin 0.05 N HCl (made in PBS) first, pH adjusted to 7.4 by NaOH, and thena stock solution of 6.5 mg/mL was prepared with PBS. The stock solutionwas then sterilized by filtering through a 0.22 μm-filter before use. Atindicated time points (4, 7, and 28 d after surgery), mice wereanesthetized with tribromoethanol solution (500-750 mg/kg), perfusedwith saline, followed by 10% neutral-buffered-formalin. Thecontralateral control (right common carotid artery) and ligated LCCAwere then carefully dissected, excised, and immersed in 10% formalinbefore processing and embedding in paraffin.

Statistical Analysis

All values were given as mean±S.D. The statistical significance ofdifference between treatment and control groups was calculated with at-test or 1-way ANOVA. P values of less than 0.05 were consideredstatistically significant

[Results] 5-Methoxytryptophan Derivatives Control COX-2 Expression.

After pretreating A549 cells with different concentrations of 5-MTP,5-MTPE and NACT-5-MTP for 30 min, cells were stimulated with PMA for 8h. Cell lysates were immunoblotted with antibodies for COX-2 or β-actin.The experiments were repeated 3 times with similar results.

According to the result shown in FIG. 1F, as 5-MTP effect, both 5-MTPEand NACT-5MTP dose-dependently blocked PMA-induced COX-2 proteinexpression in A549 cells. Furthermore, the COX-2 suppressive activity ofboth 5-MTP derivatives is potent than that of 5-MTP.

As serotonin, 5-methoxytryptamine and tryptophan share an indolebackbone with 5-MTP, we determined the effect of these compounds onPMA-induced COX-2 expressions in A549 cells. As shown in the followingTable 1, neither serotonin nor 5-methoxytryptamine or tryptophan exertedan effect on COX-2 expression levels, suggesting that the5-methoxy-indole-3-propionic acid moiety is required for inhibitingPMA-induced COX-2 expression. Collectively, these results demonstratedthat 5-MTP and its derivatives suppress PMA-induced COX-2 expression inA549 lung cancer cells.

TABLE 1 Suppressive activity in PMA-induced A549 COX-2 expressionTryptophan − Serotonin − 5-methoxytryptamine − 5-MTP + 5-MTPE +

5-Methoxytryptophan Derivatives Block Cancer Migration and Tumor Growth.

Lung cancer A549 and breast cancers BT474 and T47D cells were pretreatedwith different concentrations of 5-MTP or 5-MTPE for 30 min, thenstimulated with PMA for 4 h or 24 h. Cell migration was measured by thetranswell migration assay. The results show that 5-MTP, 5-MTPE andNACT-5MTP abrogated A549 (FIG. 2A) and breast cancer BT474 and T47D(FIG. 2B) cell migration induced by PMA for 4 h or 24 h.

In addition, A549 (5×10⁶ cells) was injected subcutaneously into theflank of SCID-Beige mice. 10 mice each received intraperitonealinjection of 5-MTP (100 mg/kg), 5-MTPE (100 mg/kg) or vehicle twiceweekly. Caliper measurement of the subcutaneous tumor volumeperiodically for 7 weeks. The result shown in FIG. 2C indicates that5-MTP and 5-MTPE suppressed tumor volume in a time-dependent manner. Theaverage tumor volume at 7-week in the 5-MTP treated group was ˜50% ofthat in the control group.

The results shown in FIGS. 2A-2C indicate the 5-MTP and its derivativesinhibit cancer migration and tumor growth in vitro and in vivo.

5-Methoxytryptophan Derivatives Inhibit LPS-Induced COX-2 Expression andCytokine Production in Macrophages.

After pretreating mouse macrophage RAW264.7 cells with differentconcentrations of 5-MTP for 30 min, cells were stimulated with LPS for 8h. Cell lysates were immunoblotted with antibodies for COX-2 or β-actin.In addition, RAW264.7 cells were transfected with COX-2promoter-luciferase plasmid. After 24 h transfection, the cells wereincubated with LPS for 8 h. Luciferase activity was measured. Theexperiments were repeated 3 times with similar results. The resultsshown in FIGS. 3A-3B confirmed that 5-MTP blocked LPS-induced COX-2protein expression and promoter activity in a concentration-dependentmanner in RAW264.7 cells.

Furthermore, peritoneal macrophages were stimulated with LPS with orwithout 5-MTP. After 8 h, COX-2 protein expression was determined bywestern blot. The experiments were repeated 3 times with similarresults. The results shown in FIG. 3C indicates that 5-MTP inhibitedLPS-induced COX-2 expression in primary mouse peritoneal macrophages.

Moreover, RAW264.7 cells and IL-6 promoter-luciferase plasmidtransfected RAW264.7 cells were pretreated with different concentrationsof 5-MTP for 30 min, followed by LPS stimulation for 8 h. IL-6 promoteractivity was measured by luciferase assay. IL-6 level in culturesupernatants was measured by ELISA. According to the results shown inFIGS. 3D-3E, 5-MTP inhibited LPS-induced IL-6 promoter activity andprotein expression in RAW264.7 cells in a concentration-dependentmanner, comparable to its inhibition of COX-2 expression (FIGS. 3A-3B).

Furthermore, peritoneal macrophages were stimulated with LPS with orwithout 5-MTP. After 24 h, proinflammatory cytokines were measured byELISA. The results shown in FIG. 3F indicates that 5-MTP inhibited IL-6,IL-1β and TNF-α production in peritoneal macrophages and TNF-αproduction in RAW264.7 cells (data not shown).

On the contrary, neither serotonin nor 5-methoxytryptamine or tryptophanexerted an effect on COX-2 and IL-6 expression levels, as shown in thefollowing Table 2.

TABLE 2 Suppressive activity in LPS-induced cytokine productionTryptophan − Serotonin − 5-methoxytryptamine − 5-MTP + 5-MTPE +

Furthermore, RAW264.7 cells were treated with different concentrationsof 5-MTP or 5-MTPE for 24 h. Cell viability was determined by MTT assay.In another experiment, after pretreating RAW264.7 cells with differentconcentrations of 5-MTP or 5-MTPE for 30 min, cells were stimulated withLPS for 24 h. IL-6 level in culture supernatants was measured by ELISA.As shown in FIGS. 4A-4B, the 5-MTP derivatives, 5-MTPE significantlyinhibited LPS-induced cytokine production but not cell viability.

From the results shown in FIGS. 3A-4B and Table 2, these results implythat the 5-methoxy-indole-3-propionic acid moiety is required forinhibiting LPS-induced COX-2 and IL-6 production. Overall, these resultsindicate that 5-MTP and its derivatives 5-MTPE suppresses LPS-inducedinflammatory responses in mouse RAW264.7 cells and peritonealmacrophages.

5-Methoxytryptophan Derivatives Protect Against Lethal Endotoxemia inMice.

Since suppression of 5-MTP by endotoxemia could contribute touncontrolled macrophage overproduction of proinflammatory cytokines andmediators, we determined whether exogenous 5-MTP administration rescuesmice from LPS-induced systemic inflammation, organ damage and mortality.Mice were injected with saline, 5-MTP (23.4 mg/kg), 5-MTPE (25 mg/kg) orvehicle for 30 min, followed by LPS (60 mg/kg) (saline, n=25; 5-MTP,n=7; 5-MTPE, n=7; LPS, n=30; LPS+5-MTP, n=25; LPS+5-MTPE, n=25).Survival was monitored during the next 72 h. *P<0.001 compared to LPStreatment.

As shown in FIG. 5A, mice treated with LPS started to die at day 1, andmore than 65% of mice died at 72 h. By contrast, none of the micetreated with 5-MTP died at day 1 and only 20% of endotoxemic mice diedat day 3 while no mortality was observed in control groups (saline or5-MTP alone). Notably, 5-MTPE showed a 100% protective capacity inLPS-induced lethal endotoxemia. To confirm the protective effect of5-MTP in endotoxemia, we used cecal ligation and puncture (CLP) model inmice. 5-MTP-afforded protection was also observed in mice challengedwith CLP. It significantly improved survival from 0% to 44.4% at day 2.This improved survival was maintained up to day 6 (FIG. 5B).

In addition, paraffin-embedded sections were prepared from lungs of miceinjected with 60 mg/kg LPS with or without saline or 5-MTP for theindicated time. Lung tissues were stained with hematoxylin and eosin andexamined under light microscopy. The results are shown in FIG. 5B, inwhich scale bars represent 100 μm.

As shown in FIG. 5B, examination of lung tissues revealed that LPSinduced time-dependent progression of polymorphonuclear leukocyteinfiltration, alveolar septal wall thickening, interstitial edema, andalveolar congestion, consistent with pathological changes of sepsis.5-MTP significantly alleviated these pathological changes caused by LPS.

Furthermore, bronchoalveolar lavage fluid (BALF) was isolated from micetreated with LPS with or without various concentrations of 5-MTP for 24h. Cell number in BALF was determined by trypan blue exclusion assay.

As shown in FIG. 5C, at 24 h after LPS administration, there was asignificant increase in total cell number in the bronchoalveolar lavagefluid (BALF), which was reduced by 5-MTP in a dose-dependent manner.

Moreover, paraffin-embedded sections were prepared from lungs of miceinjected with 60 mg/kg LPS with or without saline or 5-MTP at 12 h, 16 hand 24 h. COX-2 and inducible nitric oxide synthase (iNOS) proteinlevels in lung tissues were determined by immunohistochemistry bystaining with anti-COX-2 and anti-iNOS antibodies, respectively. Theresults are shown in FIGS. 6A-6B, in which scale bars represent 100 μm.As shown in FIGS. 6A-6B, 5-MTP suppresses COX-2 (FIG. 6A) and iNOS (FIG.6B) expression in lung tissues of LPS-induced endotoxemia, which isconsistent with the results that 5-MTP suppresses macrophage COX-2expression and cytokine productions (especially, iNOS productions).

Mice were injected intraperitoneally with or without differentconcentrations of 5-MTP or saline for 30 min, followed by LPSadministration for the indicated time. Proinflammatory cytokines levelin serum was measured by ELISA. (n=6 per group). The result shown inFIGS. 7A-7D respectively indicates that 5-MTP abated the elevation ofblood level of IL-1β, TNF-α, IL-6 and INF-κ in LPS-treated mice in adose- and time-dependent manner. Also, 5-MTP at 23.4 mg/kg significantlyreduced all the tested cytokines and at 100 mg/kg it reduced thecytokines to the basal level. In addition, 5-MTP suppressed the rise ofIL-12 in LPS-treated mice (data not shown). These results suggest that5-MTP protects against LPS-induced lung damages and mortality bypreventing cytokine, prostaglandin and NO storm.

To further confirm that 5-MTP deactivates peritoneal macrophages duringendotoxemia, we evaluated the effect of 5-MTP on IL-1β and IL-6production ex vivo in peritoneal macrophages isolated from untreated or5-MTP-treated endotoxemic mice. Peritoneal macrophages were isolated 8 hafter LPS injection with saline or 5-MTP (23.4 mg/kg) and cultured withmedium alone. After 24 h, proinflammatory cytokines were measured byELISA (n=3). As show in FIG. 7E, macrophages from untreated micespontaneously produced high amounts of IL-1β and IL-6, both of whichwere significantly suppressed by 5-MTP.

To ensure that 5-MTP administration increases serum 5-MTP, we analyzed5-MTP level at 24 h and 72 h after 5-MTP infusion. Serum 5-MTPconcentrations in mice treated with saline or LPS with or without 5-MTP(23.4 mg/kg) at 24 h and 72 h. The error bars denote mean±SD (n=3). Asshown in FIG. 7F, serum 5-MTP remained highly elevated, several-foldover the basal level at 24 h after 5-MTP administration and returned tobasal level at 72 h.

These results support the notion that 5-MTP suppression by LPScontributes to systemic inflammation, lung damage and death, andsupplement with 5-MTP to boost the level of 5-MTP confers control ofsystemic inflammation and protection against tissue damage and death.

5-MTP Reduces Neutrophil Infiltration and Chemokine Production.

Thirty min after saline or 5-MTP administration, mice were injectedintraperitoneally with LPS at 4 h, 12 h, 16 h and 24 h.Paraffin-embedded lung tissue specimens were subjected toimmunohistochemical Ly6G staining for determination of neutrophilinfiltration in lung tissues. The results are shown in FIG. 8A, in whichscale bars represent 100 μm. As shown in FIG. 8A, neutrophilinfiltration was slightly increased at 4 h and became markedly increasedat 12 h and thereafter following LPS treatment. Also, 5-MTPdose-dependently reduced neutrophil infiltration; and at 100 mg/kg, itreduced the infiltration almost to the basal level.

In addition, MPO activity was determined in lung tissues of mice treatedwith saline or LPS in the presence or absence of 5-MTP (23.4 mg/kg) for16 h (n=6 mice/group). As shown in FIG. 8B, analysis of lung MPOactivity confirmed that 5-MTP reduced neutrophil infiltration to thebasal level.

Furthermore, chemokine level was measured by ELISA in the serum ofsaline or LPS-injected mice with or without saline or 5-MTP (23.4 mg/kg)after 24 h (n=10). The results shown in FIG. 8C indicate thatLPS-induced elevation of chemokines, CXCL1, MCP-1, RANTES and eotaxin inthe serum was blunted by 5-MTP treatment at 24 h.

The results shown in FIGS. 8A-8C suggest that 5-MTP reducessepsis-mediated lung damage by suppressing macrophage activation andcytokine production as well as reducing neutrophil infiltration andmultiple proinflammatory chemokine production.

5-MTP Prevents LPS-Induced Lung and Spleen Cell Apoptosis.

Paraffin-embedded sections were prepared from lung tissues of micetreated with saline or LPS (60 mg/kg) with or without variousconcentrations of 5-MTP for 12 or 16 h. Activated caspase-3 level inlung tissues was determined by immunohistochemical staining of cleavedcaspase-3. The results are shown in FIG. 9A, in which scale barsrepresent 200 μm. As shown in FIG. 9A, LPS treatment resulted in asignificant increase in apoptosis as demonstrated by increased cleavedcaspase-3 at 12 and 16 h which was reduced by 5-MTP.

In addition, caspase-3 activity in splenocytes of mice treated withsaline, LPS or LPS plus 5-MTP (100 mg/kg) for 16 h was measured byfluorogenic substrate as described above. The results are shown in FIG.9B, in which the error bare denotes mean±SD (n=6 mice/group). As shownin FIG. 9B, 5-MTP also prevented LPS-induced caspase-3 cleavage inspleen cells.

Furthermore, spleen weight/body weight (SW/BW) ratio was also assessed(n=6 mice/group). As shown in FIG. 9C, in LPS-challenged mice, thespleen weight/body weight (SW/BW) ratio was markedly increased ascompared with that of saline-treated mice, consistent with severe spleenedema. 5-MTP administration ameliorated spleen edema in a dose-dependentmanner.

According to the results shown in FIGS. 9A-9C, these results suggestthat 5-MTP prevents LPS-induced lung and spleen cell apoptosis inendotoxemic mice.

5-MTP Suppresses LPS-Mediated Immune Signaling.

After pretreating RAW264.7 cells with different concentrations of 5-MTPfor 30 min, cells were stimulated with LPS for 4 h. Cell lysates wereimmunoblotted with antibodies for p38, phospho-p38 (p-p38), ERK1/2,phospho-ERK1/2 (p-ERK1/2) or β-actin. As shown in FIG. 10A, 5-MTPdose-dependently blocked the phosphorylation of p38 MAPK but not that ofERK1/2 in LPS-treated RAW264.7 cells. Activation of p38 MAPK inmacrophages is required for TLR-induced NF-κB activation, which is oneof the key factors affecting TLR-induced cytokine production. Thus, weevaluated the effect of 5-MTP on LPS-induced NF-κB activation.

After pretreating RAW264.7 cells with different concentrations of 5-MTPfor 30 min, cells were stimulated with LPS for 8 h. NF-κB promoteractivity was measured by NF-κB promoter-luciferase assay. As shown inFIG. 10B, 5-MTP suppressed NF-κB transactivation in aconcentration-dependent manner.

Furthermore, after pretreating RAW264 0.7 cells with differentconcentrations of 5-MTP for 30 min, cells were stimulated with LPS for 4h. NF-κB p65, phospho-NF-κB p65 (p-p65) (Ser536) or β-actin wasdetermined by immunoblotting with specific antibodies. The results areshown in FIG. 10C. Also, peritoneal macrophages were treated with LPSwith or without 5-MTP (50 μM) for the indicated time. Phospho-p65 in thelysates was analyzed by an ELISA kit. The results are shown in FIG. 10D,in which the error bars denote mean±SD (n=3). According to FIGS.10C-10D, 5-MTP decreased LPS-induced phosphorylation of NF-κB p65 notonly in RAW264.7 cells (FIG. 10C) but also in peritoneal macrophages(FIG. 10D).

Since NF-κB activation plays a critical role in the initiation andprogression of systemic inflammation and septic pathology, the effect of5-MTP on NF-κB activation in lung tissue was evaluated. Phospho-p65level in lung tissues was determined by an ELISA kit at 4 and 8 h afterLPS infusion with or without 5-MTP (23.4 mg/kg) (n=6 mice/group). Theresults shown in FIG. 10E indicate that LPS comparably increased thephosphorylation level of NF-κB p65 in lung tissues, which was reduced by5-MTP.

5-MTP Suppresses p300 Histone Acetyltransferase (HAT) Activation.

Activation of p300 HAT plays an important role in transcriptionalcoactivation of NF-κB and expression of inflammatory genes, such asCOX-2 and iNOS. Hence, we investigated the inhibition of 5-MTP on p300HAT activation.

p300 HAT activity in peritoneal macrophages treated with LPS in thepresence or absence of 5-MTP (50 μM) for the indicated time was measuredwith an p300 HAT activity assay kit. As shown in FIG. 10F, LPS activatedp300 HAT in peritoneal macrophages at 4 h and persisted for 24 h. Also,5-MTP significantly blocked p300 HAT activation up to 24 h.

In addition, p300 HAT activity in lung tissues of mice (n=6 mice/group)at 8-24 h after LPS infusion with or without 5-MTP (23.4 mg/kg) wasdetermined by an assay kit. As shown in FIG. 10G, in vivo, LPS elevatedp300 HAT activity in lung tissues but was suppressed by 5-MTP as inmacrophages in vitro.

Based on the results illustrated above, these results suggest that 5-MTPinhibits LPS-induced p300 HAT activation and NF-κB activity, therebysuppressing COX-2, iNOS and proinflammatory cytokine expressions.Collective inhibition of cytokines and proinflammatory COX-2 and iNOSaccounts for the protective effect of 5-MTP on sepsis, which isrepresented in FIG. 10H.

Suppressive Effectiveness of 5-MTP, 5-MTPE and COX-2 Inhibitors andNF-κB Inhibitor in LPS-Induced COX-2 and IL-6 Expression.

Because COX-2 inhibitors are considered to be anti-inflammatory agent,we next evaluated the suppressive effectiveness of 5-MTP derivatives andCOX-2 inhibitors, NS398 and SC560 in LPS-induced IL-6 production.

Herein, after pretreating RAW264.7 cells with different concentrationsof 5-MTP, 5-MTPE, COX-2 inhibitors (NSC398 or SC560) and NF-κB inhibitorJSH-23 for 30 min, cells were stimulated with LPS. In addition, RAW264.7cells were also transfected with COX-2 promoter-luciferase plasmid.After 24 h transfection, the cells were incubated with LPS for 8 h.Luciferase activity was measured.

FIG. 11A shows IL-6 level in culture supernatants measured by ELISA at24 h after LPS treatment. The results indicate that all used agentsdose-dependably blocked LPS-induced IL-6 production in RAW264.7 cells.In comparison between each other, 5-MTPE possesses a better suppressivecapacity on IL-6 production.

As demonstrated above, 5-MTP inhibited LPS-induced systemic inflammationvia suppressing NF-κB activation. Furthermore, NF-κB inhibitor has beenreported to be anti-inflammatory and anti-cancer agent. We were tocompare the suppressive effectiveness of 5-MTP derivatives with that ofNF-κB inhibitor JSH-23 in LPS-induced COX-2 and IL-6 expression. AsJSH-23 effect, both 5-MTP and 5MTPE dose-dependently blocked LPS-inducedCOX-2 and IL-6 production in RAW264.7 cells. Notably, the COX-2 and IL-6suppressive activity of 5-MTPE is potent than that of NF-κB inhibitorJSH-23, as shown in FIG. 11B-11C.

Serum 5-MTP Concentrations Inversely Correlate with Coronary ArteryDisease

To investigate the clinical relevance of 5-MTP in CAD, we measured serum5-MTP levels in control subjects and patients with CAD. Forty controlsubjects (26 males and 14 females, mean age 50±11 years) and 40 CADpatients (31 males and 9 females, mean age 62±12 years) were included inthe study. The presence of CAD was confirmed by coronary angiography andCAD was defined as more than 50% angiographic diameter stenosis in oneor more coronary arteries. The body mass index was not different betweencontrol and CAD groups (25.5±3.7 vs. 25.6±4.1, respectively). Controlsubjects did not have any coronary vessel and known systemic disease.Serum was obtained from blood drawn from controls or patients prior toangiography. Measurements of 5-MTP with enzyme-immunoassays revealedthat the mean serum 5-MTP levels from controls were 0.85±0.03 μmol/L(FIG. 12A). In contrast, serum 5-MTP concentrations of CAD patients weresignificantly reduced to 0.20±0.02 μmol/L (FIG. 12A, P<0.0001 vs.controls). These results indicate that serum 5-MTP concentrationsinversely correlate with coronary artery disease.

5-MTP Derivatives Protects Against Atherosclerosis and VascularCalcification in ApoE-Deficient Mice

Since reduction of serum 5-MTP in CAD patients may be associated withthe development of vascular disease, we determined whether exogenous5-MTP derivatives exert protective effect in the development ofatherosclerosis. ApoE-deficient mice were fed a Western high fat dietcontaining 1.25% cholesterol and 21% fat (Research Diets) starting at 6weeks of age. The mice were simultaneously treated with vehicle (PBS),5-MTP or 5-MTPE (23.5 mg/kg body weight, 3 times a week) byintraperitoneal injection. After 8 weeks, the aortic trees weredissected and lesions examined in the aortic arch and its branches. Ourdata from a pilot experiment indicated that the abundant whitish lipidaccumulation was readily visible in vehicle-treated mice (FIG. 12B,arrows). In comparison, 5-MTP-treated mice had less lipid accumulationin the arteries (FIG. 12B, arrows). In similar results were observed in5-MTPE-treated mice, Oil red O analysis indicated that 5-MTPE suppressedlipid accumulation in atherosclerotic lesions of ApoE-deficient micetreat with high fat diet as compared with vehicle-treated mice (FIG.12C).

Vascular smooth muscle cell (VSMC) calcification is the major phenomenonto induce atherosclerotic vascular calcification in cardiovasculardisease (CVD). We next investigated the effect of 5-MTP derivatives onVSMC calcification. Calcifying medium-induced calcification in vascularsmooth muscle cells was reduced by 5-MTP and 5-MTPE (FIG. 12D). Inaddition, 5-MTP also prevented high fat diet-induced vascularcalcification in ApoE-deficient mice (FIG. 12E).

5-MTP Reduces Neointima Formation in a Mouse Carotid Artery LigationModel

To begin to investigate the potential function of 5-MTP in vascularremodeling, we subjected mice to a neointima formation model of carotidartery ligation, and treated mice with vehicle or 5-MTP. Four weeksafter ligation, H&E and elastin staining revealed robust neointimaformation in vehicle-treated ligated carotid arteries (FIG. 13A, 13C).In contrast, intimal thickening was reduced in ligated carotids from5-MTP-treated mice (FIG. 13B, 13D). Quantitative analysis showed that 5MTP significantly decreased intima/media ratio from 0.96±0.19 of vehiclegroup (n=9) to 0.58±0.11 of 5-MTP group (n=12, P<0.05; FIG. 13E).

In conclusion, we have shown that 5-MTP and its derivatives, 5-MTPE andNACT-5-MTP are efficacious in controlling COX-2, cytokine andinflammatory mediator overproduction, accompanied by prevention ofcancer cell growth and migration, lung damages and improvement ofmortality caused by systemic inflammation. 5-MTP may exert its effectsby functioning as a circulating hormone to control excessive COX-2expression and systemic inflammation. 5-MTP and its derivatives will bea valuable drug and/or serves as a lead compound for developing newdrugs for treating cancer and inflammatory diseases such as sepsis,Systemic Lupus Erythematosus (SLE), cardiovascular diseases, metabolicsyndrome, cancer, septicemia and diverse inflammatory joint,gastrointestinal and renal diseases.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

1. A method for treating an inflammatory-related disease, comprising administering to a subject in need thereof and effective amount of a compound of formula (I):

Wherein R₁ is H, halogen atom, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₂ is C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₃ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₄ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, C(O)R_(a), or C(O)OR_(a); in which R_(a) is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; R₅ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and n is 0-5.
 2. The method as claimed in claim 1, wherein each R₁ and R₂, independently, is H or C₁-C₁₀ alkyl.
 3. The method as claimed in claim 2, wherein R₁ is H, and R₂ is C₁-C₃ alkyl.
 4. The method as claimed in claim 1, wherein R₃ is H, C₁-C₁₀ alkyl, and R₄ is H, C₁-C₁₀ alkyl, C(O)R_(a), or C(O)OR_(a), in which R_(a) is H, or C₁-C₁₀ alkyl.
 5. The method as claimed in claim 4, wherein R₃ is H, and R₄ is H or C(O)R_(a), in which R_(a) is C₁-C₃ alkyl.
 6. The method as claimed in claim 1, wherein R₅ is H or C₁-C₁₀ alkyl.
 7. The method as claimed in claim 6, wherein R₅ is H or C₁-C₃ alkyl.
 8. The method as claimed in claim 1, wherein n is 1 or
 2. 9. The method as claimed in claim 1, wherein the inflammatory-related disease is sepsis, Systemic Lupus Erythematosus (SLE), cardiovascular diseases, metabolic syndrome, cancer, septicemia and diverse inflammatory joint, gastrointestinal, or renal diseases.
 10. The method as claimed in claim 9, wherein the inflammatory-related disease is sepsis, SLE or cancer.
 11. The method as claimed in claim 1, wherein the compound is


12. A method for treating a cancer, comprising administering to a subject in need thereof and effective amount of a compound of formula (I):

Wherein R₁ is H, halogen atom, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₂ is alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₃ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₄ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, C(O)R_(a), or C(O)OR_(a); in which R_(a) is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; R₅ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and n is 0-5.
 13. The method as claimed in claim 12, wherein each R₁ and R₂, independently, is H or C₁-C₁₀ alkyl.
 14. The method as claimed in claim 13, wherein R₁ is H, and R₂ is C₁-C₃ alkyl.
 15. The method as claimed in claim 12, wherein R₃ is H, C₁-C₁₀ alkyl, and R₄ is H, C₁-C₁₀ alkyl, C(O)R_(a), or C(O)OR_(a), in which R_(a) is H, or C₁-C₁₀ alkyl.
 16. The method as claimed in claim 15, wherein R₃ is H, and R₄ is H or C(O)R_(a), in which R_(a) is C₁-C₃ alkyl.
 17. The method as claimed in claim 12, wherein R₅ is H or C₁-C₁₀ alkyl.
 18. The method as claimed in claim 17, wherein R₅ is H or C₁-C₃ alkyl.
 19. The method as claimed in claim 12, wherein n is 1 or
 2. 20. The method as claimed in claim 12, wherein the cancer is a lung cancer, or a breast cancer.
 21. The method as claimed in claim 1, wherein the compound is


22. A compound of formula (I):

Wherein R₁ is H, halogen atom, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₂ is C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₃ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₄ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, C(O)R_(a), or C(O)OR_(a); in which R_(a) is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; R₅ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and n is 0-5, with the proviso that R₁, R₃, R₄ and R₅ are not H when R₂ is C₁-C₁₀ alkyl.
 23. The compound as claimed in claim 22, wherein each R₁ and R₂, independently, is H or C₁-C₁₀ alkyl.
 24. The compound as claimed in claim 23, wherein R₁ is H, and R₂ is C₁-C₃ alkyl.
 25. The compound as claimed in claim 22, wherein R₃ is H, C₁-C₁₀ alkyl, and R₄ is H, C₁-C₁₀ alkyl, C(O)R_(a), or C(O)OR_(a), in which R_(a) is H, or C₁-C₁₀ alkyl.
 26. The compound as claimed in claim 25, wherein R₃ is H, and R₄ is H or C(O)R_(a), in which R_(a) is C₁-C₃ alkyl.
 27. The compound as claimed in claim 22, wherein R₅ is H or C₁-C₁₀ alkyl.
 28. The compound as claimed in claim 27, wherein R₅ is H or C₁-C₃ alkyl.
 29. The compound as claimed in claim 22, wherein n is 1 or
 2. 30. The compound as claimed in claim 22, which is


31. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and a compound of formula (I):

wherein R₁ is H, halogen atom, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₂ is C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₃ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, or C₃-C₂₀ cycloalkenyl; R₄ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ cycloalkenyl, C(O)R_(a), or C(O)OR_(a); in which R_(a) is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; R₅ is H, C₁-C₁₀ alkyl, C₃-C₂₀ cycloalkyl, C₃-C₂₀ heterocycloalkyl, aryl, or heteroaryl; and n is 0-5, with the proviso that R₁, R₃, R₄ and R₅ are not H when R₂ is C₁-C₁₀ alkyl. 